Influence of Charge Differences in the C‐Terminal Part of Nisin on Antimicrobial Activity and Signaling Capacity

Abstract

Three mutants of the lantibiotic nisin Z , in which the Val32 residue was replaced by a Glu, Lys or Trp residue, were produced and characterized for the purpose of establishing the role of charge differences in the C-terminal part of nisin on antimicrobial activity and signaling properties. 'H-NMR analyses showed that all three mutants harbor an unmodified serine residue at position 33, instead of the usual dehydroalanine. Apparently, the nature of the residue preceding the serine to be dehydrated, strongly affects the efficiency of modification. Cleavage of [Glu32,Ser33]nisin Z by endoproteinase Glu-C yielded [Glu32]nisin Z(1-32)-peptide, which has a net charge difference of -2 relative to wild-type nisin Z. The activity of [Lys32,Ser33]nisin Z against Micrococcus jlavus was similar to that of wild-type nisin, while [Trp32,Ser33]nisin Z , [Glu32,Ser33]nisin Z and [Glu32]nisin Z(1-32)-peptide exhibited 3 -5-fold reduced activity, indicating that negative charges in the C-terminal part of nisin Z are detrimental for activity. All variants showed significant loss of activity against Streptococcus thermophilus. The potency of the nisin variants to act as signaling molecules for auto-induction of biosynthesis was significantly reduced. To obtain mutant production, extracellular addition of (mutant) nisin Z to the lactococcal expression strains was essential.

Specific Binding of Nisin to the Peptidoglycan Precursor Lipid II Combines Pore Formation and Inhibition of Cell Wall Biosynthesis for Potent Antibiotic Activity

“…The high potency that derives from such a target-mediated poration may well explain why nisin is so much more effective against bacteria in which the docking molecule is available for binding as compared with a peptide that apparently acts in a nontargeted fashion such as magainin 2 (5). The results reported here and in previous papers on the molecular mechanisms of activity of nisin in the absence of lipid II (7,8,18,24,28,36) show that nisin can permeabilize membranes via two different mechanisms. At high concentrations (M range) pores can be formed without lipid II, i.e.…”

“…The lyophilized material was then dissolved in water and brought to pH 2 with 20% H 3 PO 4 . After acidification, precipitated material was removed by centrifugation, and the supernatant fractionated on reversed-phase C 18 HPLC under isocratic conditions (50 mM NaH 2 PO 4 , pH 5.2). The UDP-MurNAc-pentapeptide was identified by mass spectrometry.…”

“…Nisin Z and Nisin Mutants-Nisin Z (16) and prepeptide nisin Z with the N-terminal leader sequence of subtilin (17) as well as all mutant peptides (18) were produced in SPYS medium (3% sucrose, 1% peptone (Difco), 1% yeast extract (Difco), 0.2% NaCl, 0.002% MgSO 4 , and 1% KH 2 PO 4 , pH 6.8). Peptides were purified from supernatants by hydrophobic-interaction chromatography using Fractogel TSK butyl 650-M (Tosohaas), followed by preparative reversed phase-HPLC using acetonitrile and 0.1% trifluoroacetic acid as eluents.…”

“…Mutations were verified by mass spectroscopy. Nisin Z is a natural variant of nisin A that only differs in one single residue (Asn instead of His at position 27) and possesses almost identical antimicrobial properties (19,20 Mutations of the structural genes nisA or nisZ were performed by two successive polymerase chain reactions, and mutated genes were expressed in L. lactis NZ9800 as described previously (18). Nisin stock solutions were stored at Ϫ80°C in 0.05% acetic acid.…”

“…Nisin stock solutions were stored at Ϫ80°C in 0.05% acetic acid. The MIC was determined by the inhibition of growth of the indicator strains Micrococcus flavus DSM 1790 and Streptococcus thermophilus Rs in a liquid bioassay (16,18).…”